Nanodiamonds are carbon-based nanomaterials with an inert sp3 hybridized carbon core. In recent years, the scientific community has become increasingly interested in detonation nanodiamonds (NDs) because of their useful structural, mechanical, chemical, optical, and/or biological characteristics.1 NDs were first produced in Russia in the 1960s through the incomplete combustion of carbon-containing explosives.2 Detonation nanodiamonds are produced by the detonation of 2,4,6-trinitrotoluene (TNT) and Hexogen (RDX) in a closed system in the absence of oxygen. These diamond nanoparticles initially attracted the attention of the industrial world and they have been used, for example in the electrochemical coating of metals and to improve the physical properties of polymers and the shelf life of mechanical tools.3 Investigations into their nanoscale properties have recently studied certain biological applications.4,5,6 
An attractive feature of NDs is their uniform nanoscale size distribution. The primary particle size of a ND ranges from 4 to 5 nm,1,7,8 with a chemically inert diamond core and a shell comprising sp2-hydridized carbon structures.9 Various oxygen-containing functional groups are found on the surface of NDs,8,10,11 such as carboxyl, hydroxyl, lactone, anhydride, ketone and ether12 opening the potential for their conjugation with biochemical moieties. Upon oxidation, carboxylated nanodiamonds are obtained (Scheme 1). On the other hand, reduction processes introduce hydroxyl groups on the surface, as shown below in Scheme 1. Such oxidized or reduced forms of the nanodiamonds allow for variation in the possibilities of what can be grafted on the nanodiamond surface.13,14 Surface modifications of NDs can be achieved through either a physical or a covalent (for example, amide or ester bonding) interaction.

Physical adsorption of NDs has been reported using toxins,15 proteins,16,17 chemotherapeutic drugs,5,6,18 and nucleic acids.19,20 Carboxylic21,22 and nitrogen-containing23 functional groups have been grafted onto NDs using a radical generation mechanism, while alkyl-, amino-, and amino acid-functionalized nanodiamonds have been prepared through the chemical modification of fluorinated NDs with alkyl lithium, ethylenediamine, or glycine ethyl ester hydrochloride, respectively.24 NDs have also been functionalized with amino acids25 and alkyl chains26 via covalent bonding.
NDs have been found to be biocompatible with various cell lines,18,27,28 and they generally exhibit lower cytotoxicity than other carbon-based nanomaterials such as single- and multi-walled carbon nanotubes and carbon black.27,28 As such, they have been assessed for their ability to act as vectors for the intracellular delivery of drugs and biochemical molecules. It has been shown that the hydrophilic nature of NDs is responsible for improving the aqueous solubility of chemotherapeutic and anti-inflammatory drugs that have initially showed poor solubility in water.29 Chemotherapeutic drugs such as doxorubicin5 and 10-hydroxycamptothecin6 have been successfully delivered into cancer cells after being bound to NDs.
Polymer-coated NDs have been reported to improve the delivery of plasmid DNA (pDNA)19 and small interfering RNA (siRNA)29 into cellular systems. Contrary to self-assembling nanoparticles, NDs are useful in such applications because of their resistance to biological environmental changes, which could improve the overall delivery of an attached biomolecule.
Despite the promise NDs show as vectors for delivering small chemical drugs and large biotechnology products, it remains a challenge to obtain nanosized ND particles in laboratory and industrial settings owing to their strong tendency to assemble into micron-sized aggregates when dispersed in a polar liquid medium. As these aggregates have the potential to block capillaries, which could lead to toxic effects in the body,30 the maintenance of dispersion stability of NDs is an important requirement when developing drug delivery formulations.
One way in which dispersion stability can be improved is through the use of chemical modifications such as fluorination24 and biotinylation.31 These methods have been shown to reduce the size of ND aggregates from micrometer sizes to 16024 and 170 nm,31 respectively.
While a variety of mechanical disaggregation approaches have also been explored, stirred media milling and bead-assisted probe sonication were the most successful in achieving high dispersion stability and producing single-digit nanometer particle sizes.32 However, while primary-sized NDs may be attained by high-energy bead-assisted probe sonication, the potential for the contamination of samples with sonotrode material when using this technique32 is unacceptable in life science applications.
To the best of the inventors' knowledge, no previous reported studies are known which have examined the disaggregation of NDs to particles less than 50 nm in size using a simple mechanochemical technique that can be applied at a laboratory level.